1. Field of the Invention
This invention relates generally to biosensors utilizing hydrogels to measure the concentration of free analyte molecules in a fluid, particularly biosensors suitable for implantation in a patient to provide constant monitoring of a selected analyte. The invention also relates to health alarm systems in which a biosensor is connected to apparatus which alerts a patient and/or patient caretakers to deleterious changes in the levels of analyte in the patient""s body fluids or to other adverse changes in a patient""s condition.
2. Description of Related Art
During the past decade, intense effort has been directed toward the development of analyte monitoring biosensors as an aid to prevent complications of diseases such as diabetes. Development of an implantable analyte sensor that is specific and sensitive enough to precisely and continuously monitor analyte levels in vivo would be a significant advance. Such a sensor would also greatly facilitate data collection and biochemical research relating to analyte levels in patients.
Several new implantable techniques have been developed for analyte analysis such as glucose in clinical practice based on electrochemical principles and employing enzymes such as glucose oxidase (GOD) for analyte recognition. Potentially implantable analyte biosensors based on electrochemical transducers are the most highly developed, and this class of sensors can be further subdivided into potentiometric sensors, conductometric sensors, and amperometric sensors. At present, neither the potentiometric method nor the conductometric method appears to be suitable for in vivo analyte monitoring due to: (a) interference by species other than analyte in the physiological environment; (b) low sensitivity and logarithmic dependence of the signal on the analyte concentration. A linear dependence of the signal on analyte concentration is highly desirable because of the need for repeated recalibrations over time for implanted analyte sensors. However, non-linear calibration curves can be handled reasonably well using microprocessors.
The most advanced analyte sensors for in vivo monitoring are electrochemical sensors containing hydrogels in which an enzyme which generates hydrogen peroxide upon reaction with the analyte is immobilized, with an amperometric method being used to detect the hydrogen peroxide. This technique offers the possibility for a linear calibration curve. In the amperometric method, an electrode is used which produces a current proportional to the diffusional flux of hydrogen peroxide (H2O2) to the electrode surface, or, alternatively, proportional to the diffusional flux of oxygen (O2) to the electrode surface. An increase in the surrounding analyte concentration should increase the diffusional flux of analyte into the membrane and increase the reaction rate within the membrane. The increase in enzymatic reaction rate in turn should increase the local hydrogen peroxide concentration and decrease the local oxygen concentration within the membrane. This should lead to an increase in the current detected by a hydrogen peroxide-based electrode sensor, or a decrease in current as detected by an oxygen-based electrode sensor. The latter approach, based on detecting the oxygen flux, also requires a second oxygen-based electrode sensor located in a hydrogel without the enzyme. This second electrode is used as a reference.
However, amperometric sensors must overcome several hurdles before they will ever be useful for commercial in vivo monitoring. Current analyte sensor designs appear unlikely to solve these difficult problems in the near future. The first hurdle arises from electrochemical interference. The analyte (whether hydrogen peroxide or oxygen) must be the only species present which produces a current at the electrode. Hence for both oxygen-based and hydrogen peroxide-based analyte sensors, an inner membrane must be used which is permeable to the analyte but impermeable to endogenous interferents which may produce electrochemical effects. In clinical studies of the hydrogen peroxide-based sensor, a decay in sensitivity over the implant period was observed, a phenomenon which could not be explained by blockage of the sensor surface by protein. One possible explanation for the loss of sensitivity is hydrogen peroxide mediated enzyme deactivation. For the oxygen-based sensor, this can be avoided by co-immobilizing catalase with enzyme, because catalase consumes hydrogen peroxide. Fourthly, a shortage of oxygen relative to analyte can place an upper limit on the biosensor""s ability to measure analyte levels. This problem is called the xe2x80x9coxygen deficitxe2x80x9d.
In addition to the biosensors described above, hydrogels have also been used in devices developed to release insulin directly into a diabetic""s bloodstream in response to high analyte levels. In one approach, the hydrogel is constructed to have chemically immobilized pendant groups which are charged under physiological conditions (pH2 to pH10). Molecules of glucose and of a glucose-specific binding molecule (abbr. GBM; for example, concanavalin A) are also immobilized in the gel. Within the hydrogel, in a solution which contains no free glucose, immobilized glucose molecules bind to immobilized GBMs, forming what are in effect non-covalent xe2x80x98crosslinksxe2x80x99. As the hydrogel is exposed to a fluid containing free glucose, binding competition displaces immobilized glucose from GBMs, thus reducing the number of xe2x80x98crosslinksxe2x80x99. This reduction in crosslinking causes an increase in swelling of the hydrogel, due to the presence of the charged pendant moieties in the hydrogel. In effect, the hydrogel swelling increases the porosity and/or pore size between gel subunits. These insulin-delivery hydrogels also contain insulin, and the increase in pore size in turn allows insulin (a rather large molecule which does not diffuse readily through a closely-crosslinked gel matrix) to diffuse outward and be released into the patient""s bloodstream. See A. Obaidat, et al., Characterization of Protein Release through Glucose-sensitive Hydrogel Membranes, 18 BIOMATERIALS 801-806 (1997); Y. Ito, et al., An Insulin-releasing System that is Responsive to Glucose, 10 JOURNAL OF CONTROLLED RELEASE 195-203 (1989), which are expressly incorporated herein.
However, so far as we are aware, the changes in swelling force/osmotic pressure that occur in pH-sensitive competition binding hydrogels have not heretofore been recognized and exploited for the measurement of the concentration of free analyte. The prior art does not teach measurement of the analyte-induced swelling of the hydrogel as a method of measuring analyte concentrations, and it specifically does not teach the use of a transducer to measure hydrogel swelling. The use of a pressure transducer provides a measurement tool that avoids the problems encountered by electrochemical sensors.
Thus, a need exists for a biosensor that is extremely sensitive to the concentration of analyte, and also relatively free from interference, even when operating in complex media such as human blood. A need further exists for a biosensor that relies directly on change in analyte, since analyte concentration itself is a much more controlled parameter than parameters measured by electrodes. This is especially critical in implantable biosensors, because this system is relatively free from potential sources of interference. Additionally, there is a need for hydrogel-based biosensors in which the analyte-detecting process does not consume oxygen.
Accordingly, it is the object of this invention to provide such a biosensor. It is a further object to provide a general method for measuring the concentration of any analyte for which a suitable specific binding partner can be found or constructed.
The present invention comprises a hydrogel-based biosensor which measures the osmotic pressure within a hydrogel having pendant charged moieties, analyte molecules, and analyte binding partner molecules all immobilized within. In order to measure the osmotic pressure or swelling tendency of the hydrogel, the gel is confined in a rigid enclosure that has a semipermeable opening to permit osmotic contact between the test fluid (a patient""s blood or other solution) and pressure detection means are operably associated with the hydrogel for detecting the osmotic pressure or swelling tendency of the hydrogel. The device uses a competition assay, in which free analyte molecules diffusing into the hydrogel displace immobilized analyte molecules in proportion to the free analyte concentration. This displacement reduces the degree of xe2x80x98crosslinkingxe2x80x99 between immobilized analyte and immobilized analyte binding partner molecules, and, since the hydrogel also contains pendant charged moieties, reduction in xe2x80x98crosslinkingxe2x80x99 results in a change in swelling propensity, or osmotic pressure, detectable by the pressure detection means.
Thus, a biosensor of the invention comprises a polymeric hydrogel having pendant moieties that are charged under physiological conditions, an analyte binding molecule immobilized in the hydrogel and capable of binding the free analyte, analyte molecules immobilized in the hydrogel, and pressure detection means for measuring the osmotic pressure of the hydrogel. In the preferred embodiment, the pressure detection means is comprised of a diaphragm positioned such that changes in osmotic pressure within the gel cause changes in pressure exerted on the diaphragm, and a pressure transducer operably associated with the diaphragm for detecting these pressure changes. Biosensors of this invention can be designed to detect any analyte which can be immobilized within the hydrogel and for which an (immobilizable) binding partner with sufficient specificity and binding affinity can be found (see Table 1).
To derive analyte concentration readings, it is necessary to calibrate the detected pressure changes against solutions of known analyte concentration, as is commonly done for other measuring techniques. Accordingly, a further embodiment of the biosensor includes reporting means associated with the pressure detection means for reporting the data signal, and computing means operably disposed to receive the data signal and constructed to compare it to a predetermined calibration curve and to produce an analyte data signal reflecting the measured analyte concentration.
In a preferred embodiment, the reporting means is a battery powered telemeter which sends a radio data signal to a receiver operably attached to the computing means. In a further preferred embodiment, the computing means is associated with an alarm system. The computing means may be a personal computer, but in a preferred embodiment, the computing means is a microprocessor.
In a more highly preferred embodiment, the computing means contains or is operably associated with alarm means for providing an alarm signal when the analyte concentration falls outside a pre-determined acceptable range. In a further highly preferred embodiment, the biosensor unit carried in or on the patient""s body includes a GPS (global positioning system) unit.
Thus, a further invention described herein comprises biosensor-based health alarm system which provides a warning of an adverse condition detected by a biosensor to care providers at a location remote to the patient via telephone or wireless transmission means. In a highly preferred embodiment, the system includes a GPS unit and a wireless phone, thus providing monitoring and alarm coverage to the patient while travelling. The biosensor of the system may be any sensor configured to detect a critical health-related biological determinant (such as, but not limited to, the concentration of a selected analyte in the patient""s body fluid). The system may further include an automatic drug administration component which responds to the sensor by administering an appropriate amount of a drug to ameliorate the adverse effects of the change in the biological determinant.
The invention also encompasses a method of determining the concentration of free analyte in a solution. The method comprises steps of: providing a hydrogel having pendant charged moieties, analyte molecules, and analyte-specific binding molecules covalently immobilized therein; enclosing the hydrogel in a rigid structure which has at least one permeable portion available for contacting a test fluid with the hydrogel, the permeable portion constructed to permit free analyte to diffuse into the hydrogel; contacting the hydrogel sequentially with a series of calibration solutions having known concentrations of free analyte and measuring osmotic pressure in the hydrogel for each of the calibration solutions to produce a calibration curve of osmotic pressure versus analyte concentration; contacting the hydrogel with the test fluid, and measuring a resulting osmotic pressure; and comparing the resulting osmotic pressure with the calibration curve to determine analyte concentration of the test fluid.
The steps involving measuring the osmotic pressure are preferably accomplished by disposing pressure sensing means within the rigid structure and in contact with the hydrogel, the pressure sensing means producing a data signal reflective of the osmotic pressure.